The long-term goal of this project is to define the interactions within transcription elongation complexes and with regulatory proteins and nucleoprotein complexes that govern RNA synthesis by RNA polymerase. Pausing and premature termination by RNA polymerase affect expression of bacterial genes in many ways. The structure of the nucleoprotein template for transcription is an important component of these regulatory events. In some cases, the nucleoprotein structure of the template causes a requirement that specialized regulatory proteins modify the transcription complex to make it resistant to pausing and termination. Both the basic mechanisms of pausing and termination and the mechanisms by which regulatory proteins control pausing and termination depend on changes to interactions within the elongation complex that are poorly understood. A central target of these interactions is folding of the trigger loop in RNA polymerase into the trigger helices, which is required to catalyze RNA synthesis. The synergy between these regulatory events in RNA polymerase and the enzyme's contacts to the nucleoprotein template are even less well understood. To understand this synergy, the composition and structure of the nucleoprotein template in bacterial cells must be defined. Through collaboration with an expert in mass spectrometry, a new approach based on sequence-specific DNA capture and quantitative proteomics will be developed to analyze the composition and structure of the nucleoprotein template, to identify changes in nucleoprotein complexes in different environmental conditions, and to identify how nucleoprotein complexes change when interacting with transcribing RNA polymerase. Changes in the structure of nucleoprotein complexes in the bacterial nucleoid are known to play key roles in silencing of foreign DNA acquired by lateral gene transfer, in bacterial pathogenesis, and in bacterial stress responses. Thus, the project will have broad impact on key topics in human health ranging from gene flow in the human microbiome to microbial pathogenesis. It also will contribute to the development of an important new technology of DNA sequence-targeted unbiased proteomics with broad applications in biotechnology, human medicine, and both prokaryotic and eukaryotic molecular biology.